Insect pests cost the general public billions of dollars annually in losses. These losses include the expense of controlling insect pests as well as crop loss and property damage caused by the pests. Helicoverpa zea has the capacity to inflict devastating yield losses to agronomically important crops. Depending the type of plant the larva of feeds on, the pest is also known as either cotton bollworm when it feeds on cotton, corn earworm when it feeds on corn. The treatment of Helicoverpa zea is typically controlled through the use of pyrethroid and Bacillus thuringiensis (Bt) insecticides.
Heliothis virescens (tobacco budworm) is found in the Americas, from Canada to Argentina. It is very similar to the closely-related genus Helicoverpa, under which name both genera were formerly subsumed. Due to its high reproductive potential, H. virescens may cause considerable losses, especially in cotton, tobacco and soybean, but also in alfalfa, cabbage, lettuce, okra, pea, pepper, squash, tomato and many others crops. Helicoverpa armigera (cotton bollworm) is almost indistinguishable from its near relative H. zea. However, the two species have different areas of distribution. Helicaverpo armigera, also called the “Old World bollworm”, is found in parts of Europe, Asia, Africa and Australasia; Helicoverpa zea, the “New World Bollworm” in the Americas. Their host ranges are broadly similar.
Both species originate from tropical and subtropical regions, but they will immigrate over long distances into areas with temperate climates each summer. The adult insects are good fliers and are mostly active at night. H. armigera is very polyphageous, and is a pest of about 200 species. It also attacks a great number of cereal, vegetable and garden crops, among them beans, leek, zucchini, lemon, sunflower, artichoke, pigeonpea, sorghum and groundnut. Considered one of the most serious insect pests worldwide, causing huge losses due to its high reproductive potential and polyphagy. Economic damage is greatest in cotton and vegetables. In grain legumes, which are staple foods for people in many countries, up to 80% of the crop can be destroyed. Helicovepa zea (corn earworm) is pest to over 100 crop plants, the most important of which are corn, cotton and tomato. Other hosts include bean, broccoli, cabbage, eggplant, lettuce, okra, pea, pepper, soybean and watermelon. Corn earworm is found throughout the temperate and (sub)tropical parts of the Americas. It cannot overwinter successfully farther north than about 40° C. (104° F.); but being highly dispersive, it will immigrate into the northern USA and southern Canada each spring. Due to its high multiplication rate, H. zea can rapidly build up large populations, so the feeding caterpillars can sometimes cause devastating crop losses.
Chemical pesticides are the primary tools used to combat these insect pests. However, use of traditional chemical pesticides has disadvantages, including non-target effects on neutral or beneficial insects. Several microbial protein toxins such as the Cry or VIP proteins from Bacillus species (e.g., Bacillus thuringiensis) have been expressed in bacteria or plants to control plant pests (including Helicoverpa or Heliothis insect species). However, a constant exposure of the pest insects to these toxins is believed to eventually result in insect resistance development. Hence, new modes of action will be needed to control damaging plant pests such as Helicoverpa or Heliothis insects, particularly new modes of action unrelated to the toxins currently expressed in plants or chemistry applied on plants.
An approach to decrease dependence on chemical pesticides and provide for a new mode of action to reduce insect pest infestation is by causing a specific gene(s) of the target-pest to malfunction by either over expression or silencing gene expression. The silencing approach utilizes RNA interference pathways to knockdown the gene of interest via double-strand RNA. Double-strand RNA (dsRNA) induces sequence—specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). RNAi is a post-transcriptional, highly conserved process in eukaryotes that leads to specific gene silencing through degradation of the target mRNA. The silencing mechanism is mediated by dsRNA that is homologous in sequence to the gene of interest. The dsRNA is processed into small interfering RNA (siRNA) by an endogenous enzyme called DICER inside the target pest, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex (RISC), which finds and cleaves the target mRNA. The dsRNA inhibits expression of at least one gene within the target, which can exert a deleterious effect upon the target if that gene is required for normal development.
Fire, et al. (U.S. Pat. No. 6,506,559) disclose a process of introducing RNA into a living cell to inhibit gene expression of a target gene in that cell. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. Specifically, Fire, et al. (U.S. Pat. No. 6,506,559) disclose a method to inhibit expression of a target gene in a cell, the method comprising introduction of a double-stranded ribonucleic acid into the cell in an amount sufficient to inhibit expression of the target gene, wherein the RNA is a double-stranded molecule with a first ribonucleic acid strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second ribonucleic acid strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene. Furthermore, the first and the second ribonucleotide strands are separate complementary strands that hybridize to each other to form the said double-stranded construct, and the double-stranded construct inhibits expression of the target gene. In using dsRNA in controlling a target insect, one method is to engineer a baculovirus to produce a dsRNA construct in vivo as disclosed in Liu, et al. (U.S. Pat. No. 6,846,482).
Salient to Liu is contacting an insect with a recombinant baculovirus wherein a first ribonucleic acid sequence corresponds to at least a portion of at least one gene endogenous to the insect to control the insect. Given the advances made in the field of transfection efficiency and RNA interference, there is a need in the art to utilize RNA interference technology without using a baculovirus as a vector. Such a method would mediate control of a target-pest without depending on variables associated with a baculovirus, such as expression and transfection of dsRNA by the baculovirus.
To utilize RNA interference as a method to regulate gene expression for control, a specific essential gene needs to be targeted. Genes associated with neurohormones represent novel potential targets. One neurohoromone gene family is the pheromone-biosynthesis-activating neuropeptide (PBAN)/pyrokinin gene family. The PBAN/pyrokinin gene produces multiple peptides, each of which are defined by a similar 5-amino-acid C-terminal sequence (FXPRLamide) that is the active core fragment for these peptides as reported in Raina, A. K. and T. G. Kempe (1992). “Structure activity studies of PBAN of Helicoverpa zea (Lepidoptera: Noctuidae).” Insect Biochem Mol Biol 22(3): 221-225. It was subsequently determined that the five C-terminal amino acids, FXPRLamide, represented the minimal sequence required for activity as reported in Raina, A. K. and T. G. Kempe (1992) id.; Fonagy, A., L. Schoofs, et al. (1992). “Functional cross-reactivities of some locustamyotropins and Bombyx pheromone biosynthesis activating neuropeptide.” J Insect Physiol 38(9): 651-657; Kuniyoshi, H., H. Nagasawa, et al. (1992). “Cross-activity between pheromone biosynthesis activating neuropeptide (PBAN) and myotropic pyrokinin insect peptides.” Biosci Biotechnol Biochem 56(1): 167-8; and Raina, A. K. and T. G. Kempe (1990). “A pentapeptide of the C-terminal sequence of PBAN with pheromonotropic activity.” Insect Biochem 20(8): 849-851.
To date, over 200 PBAN/pyrokinin family peptides including peptides deduced from 40 species PBAN/pyrokinin genes have been identified. While it is one of the largest neuropeptide families in insects, the physiological functions of the PBAN/Pyrokinin peptides are only partially known. As such there is a need in the art to investigative whether the PBAN/Pyrokinin pathway can be used to interfere with essential developmental and/or reproductive functions of the targeted insect pests and result in abnormal development and/or lack of reproduction.
Furthermore there is a need for novel control methods that would interfere with essential developmental and/or reproductive functions of species that do not have the undesirable characteristics of traditional chemical pesticides. To that end, there is a need to develop dsRNA constructs that are engineered to interfere with essential developmental and/or reproductive functions of specific pest insects that would overcome some of the disadvantages of using traditional pesticides.